Ghrelin is an acylated peptide of 28 amino acids in length, which has been isolated in the stomach in humans in a specific cell type, namely in the so-called A-cells, which are mainly located in the oxyntic glands in the corpus and fundus. They contain an octanoyl ester attached to a serine residue (Kojima et al, Nature, 402, (1999), 656-660). Ghrelin and its analogues are known to be releasers of growth hormone in animals and man. These peptides act via a 7-transmembrane G-protein coupled receptor which is present both in the hypothalamus and in the pituitary (Smith et al Endocr Rev 18, (1997), 621-45).
The story of ghrelin, its receptor and synthetic compounds acting through this receptor unravelled in a unique “reverse” order. In the eighties a synthetic hexapeptide from a series of opioid-like peptides was found to be able to release growth hormone (GH) from isolated pituitary cells (Bowers C Y, Momany F, Reynolds G A, Chang D, Hong A, Chang K 1980 Structure-activity relationships of a synthetic pentapeptide that specifically releases growth hormone in vitro. Endocrinology 106:663-667). Since this action was independent of the growth hormone releasing hormone (GHRH) receptor, several pharmaceutical companies embarked upon drug discovery projects based on this hexa-peptide GH secretagogue (GHS) and its putative receptor. Several series of potent and efficient peptide as well as non-peptide GH secretagogues were consequently described in the mid nineties (Bowers C Y, Momany F A, Reynolds G A, Hong A 1984 On the in vitro and in vivo activity of a new synthetic hexapeptide that acts on the pituitary to specifically release growth hormone. Endocrinology 114:1537-1545; Patchett A A, Nargund R P, Tata J R, Chen M H, Barakat K J, Johnston D B, Cheng K, Chan W W, Butler B, Hickey G, 1995 Design and biological activities of L-163, 191 (MK-0677): a potent, orally active growth hormone secretagogue. Proc Natal Accad Sic USA 92:7001-7005; Smith R G, Cheng K, Schoen W R, Pong S S, Hickey G, Jacks T, Butler B, Chan W W, Chuan L Y, Judith F. 1993 A nonpeptidyl growth hormone secretagogue. Science 260:1640-1643).
Several years later, the receptor through which these artificial GH secretagogues acted was eventually cloned and shown to be a member of the 7TM G protein coupled receptor family (Howard A D, Feighner S D, Cully D F, Arena J P, Liberator P A, Rosenblum C I, Hamelin M, Hreniuk D L, Palyha O C, Anderson J, Paress P S, Diaz C, Chou M, Liu K K, McKee K K, Pong S S, Chuan L Y, Elbrecht A, Dashkevicz M, Heavens R, Rigby M, Sirinathsinghji D J, Dean D C, Melillo D G, Van Der Ploeg L H, 1996 A receptor in pituitary and hypothalamus that functions in growth hormone release. Science 273:974-977; Smith R G, Van Der Ploeg L H, Howard A D, Feighner S D, Cheng K, Hickey G J, Wyvratt M J, Jr., Fisher M H, Nargund R P, Patchett A A 1997 Peptidomimetic regulation of growth hormone secretion. Endocr Rev 18:621-645) In 1999 the endogenous ligand for this receptor, the hormone ghrelin, was finally discovered (Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K 1999 Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402:656-660). The main site for ghrelin production is the stomach, where the peptide is found in classical endocrine cells in the gastric mucosa. Ghrelin is reported to locally act in the gastrointestinal tract, in particular the stomach, since it has been found to stimulate gastric acid production and gastrointestinal motility (Asakawa et al., Gastroenterology 120 (2001), 337-345; Masuda et al., Biochem Biophys Res. Commun. 276 (2000), 905-908).
It is known that total gastrectomy of humans reduces circulating ghrelin levels to about 30% of those in normal individuals (Ariyasu et al., J. Clin Endocrinol Metab 86 (2001), 4753-4817). In rats, the ghrelin levels in circulation were lowered to about 20% of normal after surgical removal of the acid producing rumen and fundus parts of the stomach (Dornonville de la Cour et al., Regul. Pept. 99 (2001), 141-150).
Even though ghrelin has been found to be an appetite stimulatory signal, it has been noted that this stimulatory effect is lost after vagotomy (Asakawa, supra), giving rise to the notion that for a proper action of ghrelin the presence of the stomach is required.
In contrast to the general belief, the present inventors have now found that ghrelin and its analogues are still effective in gastrectomized individuals, where potential local effects in the stomach do not operate. These findings are surprising as it has been reported that the stimulation of feeding and hypothalamic NPY expression by ghrelin analogues is dependent on intact vagal innervation (Asakawa, supra).
The present invention also embraces the use of ghrelin analogues. In the context of the present application, analogues to ghrelin are to be understood as any peptide or non-peptide compound that essentially exerts the same biological effect as ghrelin in vivo. Exemplary non-peptide ghrelin analogues are described in EP 0 869 974 and EP 1 060 190, which illustrate a number of ghrelin analogues and which documents are incorporated herein by way of reference.
According to the present invention ghrelin may be utilized as the well-known acylated 28 amino acid peptide and may be produced by chemical synthesis or recombinant techniques. Techniques for producing peptides and linking an octanoyl ester to the serine no. 3 are well within the technical person's skill. Alternatively any of the analogues mentioned in the documents referred to above may be utilized. Preferred compounds are the compounds designated as NN 703 [5-Amino-5-methylex-2-enoic acid N-methyl-N-((1R)-1-(methyl-((1R)-1-(methylcarbamoyl-2-phenylethylcarbomoyl)-2-(naphtalen-2-yl)ethyl)amide] and MK677 [sometimes also designated MKO677, cf. Drug Discovery Today, vol. 4, No. 11, November 1999, 497-506] or NNC 26-1291, or NNC 26-1187 are growth hormone secretagogues of a non-peptidyl described in WO 99/58501 and WO 00/26252, respectively, all of which documents are incorporated herein by way of reference.
In summary, the present invention provides a method for chronic treatment of body weight loss, fat mass loss etc., occurring after gastrectomy, wherein a pharmaceutically effective amount of a substance, that upon administration to a gastrectomized patient leads to an increased level of a ghrelin receptor agonist, is administered to a patient.
Preferably, the treatment comprises increasing the appetite in the individual and increasing the body fat mass of the individual, which will eventually improve the sense of well being and the quality of life in the individual.
In addition, ghrelin and/or its analogues may also be utilized in combination with another stomach derived factor, so as to improve the results observed. Exemplarily mentioned factors are pacreastatin, gastrin, histamine, resistine, prostaglandins such as prostaglandin E2 and intrinsic factor.
In addition, ghrelin and/or its analogues may be used in combination with another body weight and body fat inducing factor. Exemplarily mentioned factors are melanin-concentating hormone (MCH), MCH receptors agonists, especially MCH receptor 1 agonists, neuropeptide Y (NPY), NPY receptor 1 agonists, NPY receptor 5 agonists, and NPY receptor 2 antagonists including peptide YY (PYY) and PYY (3-36), alpha-melanocyte stimulating hormone (alpha-MSH, alpha-melanocortin), melanocortin-3 receptor (MC3R) antagonists, melanocortin-4 receptor (MC4R) antagonists, agouti-related peptide (Agrp), Agrp-agonists, cocaine- and amphetamine-regulated transcript (CART) antagonists, orexin receptor 1 and receptor 2 agonists, growth hormone (GH), GH receptor agonists, insulin-like growth factor-1 (IGF-1), and IGF-1 receptor 1 agonists
Furthermore, the invention relates to a composition comprising ghrelin or an analogue thereof for treating malnutrition in an gastrectomized individual, in particular for increasing his appetite, body weight, specifically his body fat mass, and eventually his well being. For instance, a compound that can increase fat mass can be identified based on its ability to stimulate incorporation of glucose into triglycerides in fat cells in vitro.
The composition will contain the active ingredient together with a pharmaceutically acceptable carrier or diluent, which will be selected by the skilled artisan according to the route of administration. The pharmaceutical carrier or diluent employed may be a conventional solid or liquid carrier, e.g. lactose, cyclodextrin, talc, gelatin, agar, pectin, magnesium stearate, cellulose-derivatives, or syrup, olive oil, phospholipids, polyoxyethylene or simply water. Similarly, the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or admixed with one or more waxes. The compositions may appear in conventional forms, such as capsules, tablets, aerosols, solutions, suspensions or topical applications.
For the present indication the dosage will vary depending on the compound employed and the mode of administration. Dosage levels will vary between about 0.01 μg/kg body weight to 10 mg/kg body weight daily, preferably between about 0.01 μg/kg body weight to 1 mg/kg body weight, more preferably between 0.1 to 10 μg/kg body weight. The route of administration may be any route which effectively transports the active compound to the appropriate or desired site of action, such as oral, nasal, pulmonary, transdermal or parenteral, the oral or pulmonar route being preferred.
The objective compounds may be administered as a pharmaceutically acceptable acid addition salt or, where appropriate, as a alkali metal or alkaline earth metal or lower alkylammonium salt. Such salt forms are believed to exhibit approximately the same order of activity as the free base forms. Suitable dosages may range from about 50 mg to about 200 mg, preferably from about 20 mg to about 100 mg of the compounds of formula I admixed with a pharmaceutically acceptable carrier or diluent.